In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons, and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene;it is an idiosyncratic complication of some medical drug use;it occurs as a rare event in pregnancy and following seronegative hepatitis;and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. In the last year, we completed enrollment to our main clinical protocol for treatment of recent onset severe aplastic anemia. The study was initiated as a triple randomization among horse ATG (standard therapy in the United States for many years);rabbit ATG (newly introduced into the American market and used interchangeably with hATG);and Campath, a monoclonal antibody directed against CD52 with broad specificity to lymphocytes. The third arm was discontinued due to a low response rate after the treatment of about a dozen patients (see below). Entry into the hATG versus rATG protocol continued to completion, with 60 patients in each arm. The results of this study will be made public at the American Society of Hematology Meetings in December, when data analysis is complete, but they are likely to show marked and unexpected differences in hematologic response rates and possibly survival for two agents that have been employed indiscriminately for this disease - - therefore likely to change practice world-wide. Second, Campath, a highly immunosuppressive monoclonal antibody, has been employed in a variety of settings. The drug has been administered to 27 aplastic anemia patients refractory to hATG therapy;Campath appeared to be equivalent to rATG in inducing hematologic responses, in about 30% of refractory patients and >50% of relapsed patients. However, only 3 of 16 patients with therapy-nave recent onset severe aplastic anemia responded, and Campath was judged to be inferior to standard hATG and therefore abandoned in this setting. The Branch has also pioneered another monoclonal antibody, daclizumab, which is directed to the interleukin 2 receptor of T cells. Long-term outcomes in patients with moderate aplastic anemia and pure red cell aplasia were reported: 7/28 moderate aplastic anemia patients achieved long-term hematologic recovery, and 38% of patients with pure red cell aplasia showed long-term transfusion independence. The toxicity profile for dacluzimab is excellent;unfortunately, the monoclonal antibody is no longer produced for the American market. In other clinical studies, retrospective examination of mortality in severe aplastic anemia has disclosed a marked decrease in deaths overall, especially in patients who failed to respond to an initial course of hATG. Multi-parameter analysis suggested that this improvement was mainly due to the introduction of superior anti-fungal therapies, their greater effectiveness and earlier implementation in severely neutropenic patients. Additionally, patients have been retreated with immunosuppression and referred earlier and more often to stem cell transplant due to decreased risks from this procedure. Also reported from the Clinical Center was the effectiveness of granulocyte transfusions in the setting of severe aplastic anemia;this product has been life-saving in patients who were severely neutropenic but not yet responded to ATG. Laboratory investigation of aplastic anemia has exploited several different modalities. We have created a mouse model of immune-mediated bone marrow failure, based on runt disease: lymph node cells from a parental strain are infused into F1 hybrid animals, resulting in rapid destruction of the host bone marrow, pancytopenia, and death from infection and bleeding. This model is dependent on lymphocytes, type 1 cytokines, and a potent innocent bystander effect. In recent work, we have demonstrated that T-bet, an important transcription factor that polarizes T-cells to type 1 cytokine production, and dysregulation of which has been implicated in human aplastic anemia, also plays a role in this animal model, as demonstrated in genetically defective animals. Lymph node cells from T-bet knock-out animals failed to induce bone marrow failure, although other cytokines were increased and associated with mild blood count abnormalities. In other murine experiments, the perforin/granzyme pathway was shown to be less important than the Fas/FasL pathway in inducing bone marrow cell destruction. Aplastic anemia in men and mouse was also addressed in imaging studies. We developed a novel methodology employing confocal microscopy to examine intact bone marrow to unprecendented depths, approximately 200 microns, in combination with multicolor antibody or flourochrome staining. This method allows digital reconstruction of the three dimensional architecture of a tissue. Confocal imaging reveals marked expansion in the number and size of fat cells in both experimental immune mediated bone marrow failure in the mouse and in humans with aplastic anemia, as well as rapid invasion of the murine bone marrow by CD8 effector T-cells. Imaging also allows visualization of the boney and vascular architecture of the marrow and of engraftment over time after marrow transplantation in animals. Aplastic anemia has also been investigated utilizing conventional and advanced flow cytometric methods. We have characterized the role of T-helper type 17 (Th17) lymphocytes, which have been associated with other autoimmune diseases. These cells are increased in patients with aplastic anemia at presentation and their presence correlated with disease activity;furthermore, Th17 and CD4 regulatory T-cells were inversely correlated. Th17 cell numbers also rise in the lymph node infusion model of bone marrow failure, although in the mouse model they appear to be of secondary importance to type 1 cytokine lymphocytes. We have also utilized multiplex cytokine analysis to examine bone marrow failure. When many dozens of cytokines levels are determined in serum or plasma, distinct signatures appear for aplastic anemia and a diagnostically confusing entity, hypocellular myelodysplastic syndrome. Multiplex cytokine analysis has also been applied to serum sickness, and certain cytokines correlate both with the concentrations of human antibody directed against xenoprotein and with the occurrence of clinical serum sickness. Certain cytokines are only produced in the presence of adequate platelet and megakaryocyte numbers, and in vitro, these cells can be shown to be the source of the proteins.